Abstract: The research aims to study the effects of different stimulants on the activation of human lymphocytes. Human peripheral blood mononuclear cells were prepared by density centrifugation. The blood's sample was provided by National Institutes for Food and Drug Control and approved by its Ethics Committee. The expressions of CD69 in CD3⁺CD4⁺ and CD3⁺CD8⁺ human T cells were detected by flow cytometry after administrated with CD3/CD28 antibody, phytohaemagglutinin (PHA), Staphylococcus auresus enterooxin B (SEB), interleukin (IL27) and PMA plus ionomycin for 24 h. The proliferation of lymphocyte was detected by CellTiter-Glo kit. The secreted IFNγ in supernatant of medium was examined by ELISA kit. The proliferation of lymphocytes had no change after exposed of CD3/CD28 antibody, SEB, IL27 and PMA plus ionomycin for 24 h. However, the CD69 expressions in CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells and IFNγ productions were significantly increased by CD3/CD28 antibody, SEB, IL27 and PMA plus ionomycin at 24 h, indicating that CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were activated under above-mentioned stimulated condition. CD3/CD28 antibody, SEB, IL27 and PMA plus ionomycin were valid stimulants for T cell activation.